1. Field of the Invention
This invention concerns a radioimmunoassay for erythropoietin. It also concerns a kit containing reagents to be used in said radioimmunoassay.
Erythropoietin is a glycoprotein hormone synthesized in the kidney which acts in the bone marrow to stimulate the production of red blood cells (erythrocytes). Its measurement in human blood is important in the evaluation and differential diagnosis of certain anemias and is also useful in following renal transplant patients. However, the only reliable assay in use today has been a bioassay in which mice are made polycythemic by exposure to hypoxia in special chambers of hypertransfusion and the rate of incorporation of .sup.59 Fe into newly formed red cells is measured. This assay is not sensitive and practical for routine use and only a small number of research laboratories have developed the bioasay capability.
The earliest in vivo assays for erythropoietin consisted of the measurement of increases in hemoglobin, hematocrit, marrow erythroblasts and blood reticulocytes in normal animals. These techniques were soon replaced by the measurement of .sup.59 Fe incorporation into newly formed red cells following the administration of plasma to normal rats. See L. T. Plzak et al., J. Lab. Clin. Med. 46:671, 1955. Once the ratio of oxygen supply to oxygen demand was recognized as the controlling signal for erythropoetin production sensitive test animals in which endogenous erythropoietin production has been suppressed by decreasing tissue oxygen demand were used in erythropoietin assays. Various methods of decreasing tissue oxygen demand have been used. For example fasting, see Fried et al., Proc. Soc. Exp. Biol. Med. 94:237 (1957); increasing tissue oxygen supply by the induction of polycychemia with transfusion, see Jacobson L. et al., Proc. Soc. Expl. Biol. Med. 94:243 (1957 and the induction of hypoxia, see Cotes et al., Nature 191:1065-1067 (1967).
In these assays a dose of 0.5 to 1.0 International Reference Preparation (IRP) units of erythropoietin in starved rats and 0.05 to 0.1 units in polycythemic mice were necessary to induce a detectable elevation in the precent .sup.59 Fe incorporation in newly formed red cells.
Because of the high cost, relative insensitivity and the large amounts of material needed for the in vivo assay for erythropoietin, attempts have been made to develop better in vitro assays for erythropoietin. These methods have included measuring .sup.59 Fe incorporation heme in marrow cultures, see Krantz et al., J. Biol. Chem. 238:4085 (1963); double immuno diffusion, Krugers et al., Ann: N.Y. Acad. Sci. 149:294 (1968); hemagglutination, Lange et al., J. Lab. Clin. Med. 73:78 (1969), and radioimmunoassay, Fisher et al., "Factors Influencing Renal and Extra Renal Erythropoietin Production", In "Regulation of Erythropoiesis and Haemoglobin Synthesis", (T. Travnicek and Jan Neuwirt eds.), Proceedings of the International Symposium, University Karlova, Praha, 1971, pp. 23-40., Lertora et al., "Studies on Radioimmunoassay for Human Erythropoietin", J. Lab. Clin. Med., 86:140-151 (1975), and Garcia, J. F. "The Radioimmunoassay of Human Plasma Erythropoietin", In Regulation of Erythropoiesis, Gordon A. S. et al., eds., First International conference on Hematopotesis, capri, Italy, 1971, Milano, Italy, 1972, the Publishing House 1 Ponte, pp. 132-153. The marrow culture method has the disadvantage that in order to assay plasmas with low concentrations of erythropoietin, large volumes of plasma must be added to the in vitro medium. Since it contains interfering components the plasma sample becomes the major constituent making it difficult to interpret the results.
Radioimmunoassay has several advantages over the bioassay with regard to sensitivity, specificity, precision and practicality. The basic principle of this procedure is the competition between labelled and unlabelled antigen for a fixed number of antibody binding sites as outlined in FIG. 1. If increasing amounts of unlabelled antigen (i.e. standards or unknowns) and a fixed amount of tracer are allowed to react with a constant and limiting amount of antibody, a decreasing quantity of labelled antigen is bound to the antibody. The bound antigen is separated from free antigen by a suitable technique and percent labelled antigen bound is determined by gamma counting. The relationship of bound labelled antigen to the added non-labelled antigen is expressed as a standard curve and the amount of antigen in the sample is determined by interpolation from this curve. Iodine.sup.125 is the most widely used isotope to label proteins. It is preferred to C.sup.14 and tritium because of its high specific activity and ease of labelling and counting. It is better than Iodine.sup.131 because of its longer half-life. We attempted several iodination techniques to label erythropoietin. These methods included the Chloramine-T method of Greenwood and Hunter (4), the gaseous diffusion method of Butt (5), the microelectrolytic method of Donnabein (6 ), and the enzymatic iodination method of Marcholonis (7) and David and Reisfeld (8). None of these methods in our hands gave us efficient labelling and the majority of the labelled erythropoietin lost its immunoreactivity after iodination.
The advantages of radioimmunoassay in the identification of erythropoietin have been appreciated, see Fisher et al., I, A Radioimmunoassay for Human Urinary Erythropoietin, Israel Journal of Medical Science, Vol. 7, No. 7-8, pp. 873-876 (July-August, 1971). Fisher et al., II; "Studies on Radioimmunoassay of Human Urinary Erythropoietin", Abstract for 3rd International Congress of Hematology, Munich, Germany, Aug. 28, 1970; Fisher et al., III, "Factors Influencing Renal and Extra Renal Erythropoietin Production", In: Regulation of Erythropoiesis and Hemoglobin Synthesis, T. Travnicek and Jan Newwirt eds., Proceedings of the International Symposium, Universita Karlova, Praha, 1971, pages 23-40, Fisher et al., IV. "A Radioimmunoassay for Human Urinary Erythropoietin", Israel J. Med. Sci., 7:873-876 (1970), Lertora et al., I; "A Radioimmunoassay of Erythropoietin in Serum from Normal and Anemia Uremic Subjects"; Federation Proceedings, 32:872 (1973); Lertora et al., II, "Studies on Radioimmunoassay for Human Erythropoietin", J. Lab. Clin. Med., 86:140-151 (1975).
Fisher et al. I, II, III and Lertora et al., I and II all disclose (1) methods of purifying erythropoietin obtained from the urine of patients with anemia associated with hookworm infestation and (2) radioiodination of Erythropoietin utilizing the Chloramine T labelling method of Greenwood and Hunter. Lertora et al., II, page 149, lines 9 through 11, mention that the use of microelectrolytic procedures, conjugation labelling and enzymatic iodination with lactoperoxidase may reduce the degree of protein damage in the labelling procedure.
Except for the conjugation method, using a Bolton-Hunter reagent of the appropriate specific activity, all of the above labelling methods in our hands, have resulted in immunologically inactive labelled erythropoietin or labelled erythropoietin of low activity.
We have labelled purified erythropoietin preparations (8,000-12,000 units/mg protein) with iodine-125 using the Chloramine-T method of Greenwood and Hunter. The majority of the labelled protein obtained by this method was immunologically unreactive, probably as a result of the strong oxidizing property of Chloramine-T.
In order to circumvent the use of strong oxidizing and reducing agents, we iodinated erythropoietin by the microelectrolytic method. This method also resulted in immunologically unreactive labeled erythropoietin. In a search for a better labelling method we have also used the enzymatic lactoperoxidase method for erythropoietin iodination. Since the enzyme lactoperoxidase has a molecular weight of 84,000 and since self-iodination of the enzyme does occur during iodination, separating the enzyme from the labelled erythropoietin is difficult by gel filtration chromatography. The enzyme was therefore coupled to Sepharose 4-B and the coupled enzyme was used to catalyze the iodination reaction. Unreacted iodide was separated from the labelled erythropoietin by fractionation on a Sephadex G-25 column. The pooled protein peak from G-25 was further fractionated on Sephadex G-150. Again two labelled protein peaks were obtained. The minor protein peak appeared in the void volume and had 45-67% maximum binding to the antibody. The major labelled protein peak exhibited about 35% binding. Results obtained using free lactoperoxidase and erythropoietin were not very different from that obtaned with the solid state lactoperoxidase. The lactoperoxidase method was therefore an improvement over the Chloramine-T, and electrolytic methods, however, the labelling efficiency was again only fair and maximum binding was still low.
The Bolton-Hunter method has been used before in an attempt to label erythropoietin, See Rege et al., "Iodination Methods for The Radioimmunoassay of Erythropoietin," 17th Annual Meeting American Soc. Hematology; Atlanta, Ga., Dec. 7 through 10, 1974, p. 78. However, Rege et al. I concluded after experimenting with several methods of labelling erythropoietin that the Chloramine-T method was superior to the others when labelling less than 5 microgram quantities of erythropoietin. Rege et al. II, in 1977, discloses the use of an erythropoietin-conjugate that had been purified by absorption with antiserum to normal human urinary proteins and (2) absorption with anti-erythropoietin antiserum. However this technique fails to yield a tracer suitable for use in a practical and routine RIA.
The difficulty of labelling erythropoietin and, still retaining its biological activity was recognized recently in the development of a procedure for purifying erythropoietin, See Miyake et al., "Purification of Human Erythropoietin," The Journal of Biological Chemistry, Vol. 252:5558 (1977).
It is the primary object of this invention to provide a rapid text and reagents used therein for determining the level of erythropoietin in human sera.